1. Field of the Invention
The present invention relates to a method of designing primers for amplification of a target nucleic acid and a method of detecting the target nucleic acid by using the primer, and an assay kit for the method.
2. Description of the Related Art
For detection a nucleic acid chain having a particular nucleotide sequence, the nucleic acid is amplified by using a nucleotide primer complementary thereto. The amplification product is generally identified by electrophoresis or hybridization with a probe complementary to the sequence to be detected in the amplification product. The hybridization with a probe has the advantage that it is possible to reconfirm the specificity of amplification product.
A polymerase chain reaction (PCR) method is generally known as the gene amplification method (see, e.g., Science, 230, pp. 1350-54, 1985). However, the method has problems such as demand for complicated temperature control unit and low specificity to templates with mutation only of several bases.
Recently, a loop-mediated isothermal amplification (LAMP) method has been developed as a gene amplification method demanding no such complicated temperature control (see JP No. 3313358). The LAMP method is a method of amplifying a particular gene region under an isothermal condition at 60 to 65° C. The LAMP method use primers including an inner primer pair, an outer primer pair, and optionally loop primer pairs (see WO No. 02/024902), a strand-displacing polymerase, and a substrate nucleotide. The LAMP method gives a greater amount of final product than the PCR method. It also has advantages such as simple operation, high speed, and low cost. Accordingly, the LAMP method is expected to be used in wider fields.
The PCR products are present in the double-stranded chain structure. For this reason, when the PCR product is detected with a probe nucleic acid, its complementary chain of the PCR product inhibit as a competitor to the probe, so hybridization efficiency has decreased. To solve the problem, developed was a method of preventing self hybridization of the nucleic acid to be detected by binding a nucleic acid chain to a target nucleic acid chain in the region excluding the sequence region complementary to the probe thereof (see, e.g., JP-A 6-70799 [KOKAI]). However, even the method does not give sufficient sensitivity. Alternatively developed was a method of decomposing or separating the complementary chain in the PCR product. However, the method also had problems such as high cost due to use of enzyme, magnetic beads and the like and complicated operation.
On the other hand, the LAMP product has a single-stranded loop region therein. Therefore, it is possible to design the region to bind with a probe. In this way, it is possible to hybridize a probe with the product efficiently without a step of converting the product into single strands. Disclosed were various methods of detecting a LAMP amplification product by using a single-stranded loop region. For example, JP-A 2002-272475 (KOKAI) discloses a method of labeling a probe hybridizing with the single-stranded loop region with fluorescent dye and measuring its fluorescent polarization. Alternatively, JP-A 2002-345499 (KOKAI) discloses a method of immobilizing the 5′ terminal of a primer hybridizing with a single-stranded loop region on an immobilization carrier and monitoring its coagulation reaction. Yet alternatively, JP-A 2005-143492 (KOKAI) discloses a method of immobilizing a probe hybridizing with a single-stranded loop region on a solid phase and detecting the hybridization between the probe and the LAMP amplification product based on the fluorescent or electrochemical principle.
However, for the detection methods using a single-stranded region, there is no principle of design regions of primers and detection regions closed each other. Thus, there exists a need for an improved detection method for detecting the LAMP-amplified product efficiently.